Integrated UPLC-MS and Network Pharmacology Approach to Explore the Active Components and the Potential Mechanism of Yiqi Huoxue Decoction for Treating Nephrotic Syndrome.

Background: Yiqi Huoxue Decoction (YQHXD) is a traditional Chinese medicine that promotes blood circulation, removes blood stasis, facilitates diuresis, and alleviates edema. It is composed of 10 herbal medicines and has extensive application in treating nephrotic syndrome (NS). However, the active components and the potential mechanism of YQHXD for treating NS remain unclear. Methods: We set up a sensitive and rapid method based on Ultra-High Performance Liquid Chromatograph-Mass (UPLC-MS) to identify the compounds in YQHXD and constituents absorbed into the blood. Disease genes were collected through GeneCards, DisGeNET, and OMIM database. Genes of compounds absorbed into blood were predicted by the TCMSP database. We constructed Disease-Drug-Ingredient-Gene (DDIG) network using Cytoscape, established a Protein-protein interaction (PPI) network using String, Gene biological process (GO), and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis was performed using DAVID. Cellular experiments were performed to validate the results of network pharmacology. Result: A total of 233 compounds in YQHXD and 50 constituents absorbed into the blood of rats were identified. The 36 core targets in the PPI network were clustered in the phosphatidylinositol 3 kinase-RAC serine/threonine-protein kinase (PI3K-AKT) and nuclear factor kappa-B (NF-κB) signaling pathways. Luteolin, Wogonin, Formononetin, and Calycosin were top-ranking components as potentially active compounds. Conclusion: The results of our studies show that YQHXD is able to enhance renal function, alleviate podocyte injury, and improve adriamycin nephrotic syndrome.

Identification of the metabolites of Ixerin Z from Ixeris sonchifolia Hance in rats by HPLC-LTQ-Orbitrap mass spectrometry.

Ixerin Z, one of major sesquiterpene lactones from Ixeris sonchifolia Hance, was considered to be a major active compound because of their special structure and activity. However, studies on Ixerin Z metabolism have rarely been reported. This study is the first to investigate the in vivo metabolism of Ixerin Z following intravenously administered of Ixerin Z by HPLC-LTQ-Orbitrap mass spectrometry and multiple mass defect filters (MMDF) technique. A total of 41 metabolites as well as parent drug after using two MMDF filter templates were unambiguously or tentatively identified based on accurate mass measurements, fragmentation patterns, and chromatographic retention times. The metabolic pathways of Ixerin Z were also proposed for the first time. The results demonstrated that Ixerin Z underwent extensive metabolic reaction including hydrogenation, hydroxylation, hydrolysis, methylation, cysteine conjugation, glutathione (GSH) conjugation, sulfate conjugation, N-acetylcysteine conjugation, and glucuronidation. In conclusion, our study provided an insight into the metabolism of Ixerin Z.

A LC-MS-based method for quantification of biomarkers from serum of allergic rats.

Allergies are highly complex disorders with clinical manifestations ranging from mild oral, gastrointestinal, recurrent wheezing, and cutaneous symptoms to life-threatening systemic conditions. The levels of arachidonic acid, eicosanoids, histamine, organic acids and valine are considered to have a variety of physiological functions in connection with allergies. In this research, we have developed a RP-LC/MS method to separate and quantitate six different potential endogenous biomarkers, including leukotriene B(4) (LTB(4)), prostaglandin D(2) (PGD(2)), arachidonic acid (AA), histamine (HI), lactic acid (LA) and valine (VAL), from serum of rats with ovalbumin (OVA)-induced allergy and normal rats, and the discrepancies between the model group and the control group were compared. The separation was performed on a Prevail C18 column (250 mm x 4.6 mm, 5 microm) with a gradient elution of acetonitrile with 0.1% formic acid (v/v) and 10 mM ammonium formate (adjusted to pH 4.0 with formic acid) at a flow rate of 0.5 mL min(-1) The method was validated and shown to be sensitive, accurate (recovery values 76.16-92.57%) and precise (RSD < 10% for all compounds) with a linear range over several orders of magnitude. The method was successfully applied to rat serum and shown to be indicative of the endogenous levels of biomarkers within the rat body. The analysis of the biomarkers can provide insight into the allergic mechanisms associated with related diseases.

[Identification of dihydroflavonol glycoside isomers in Smilax glabra by HPLC-MS and HPLC-1H NMR].

OBJECTIVE: To identify dihydroflavonol glycoside isomers in Smilax glabra. METHOD: The sample was analyzed by HPLC-MS in combination with HPLC-1H-NMR. RESULT: Four dihydroflavonol glycoside isomers were identified as astilbin, neoastilbin, isoastilbin, neoisoastilbin. CONCLUSION: The method is simple and rapid for the identification of dihydroflavonol glycosides in S. glabra.

[Qualitative and quantitative determination of the main components of huanglianjiedu decoction by HPLC-UV/MS].

AIM: To establish a comprehensive HPLC analytical method of Huanglianjiedu decoction. METHODS: This study was performed by HPLC-UV/MS to identify the chemical constituents of the whole and individual herbs of the "Huanglianjiedu decoction". Zorbax Extend C18 (150 mm x 4. 6 mm ID, 5 microm) column was used; the mobile phase was composed of acetonitrile (A) and water (B, with 0.5% acetic acid) with gradient elution; the flow rate was 1.0 mL x min(-1) and the column temperature was setup at 25 degrees C. The detection wavelength was 254 nm. RESULTS: The chromatogram of Huanglianjiedu decoction showed 21 main peaks. Peaks 1, 2, 5 and 18 were from Gardenia jasminoides Ellis, Peaks 8, 13, 14, 15, 16, 17, 19 and 21 from Scutellaria baicalensis Georgi. While 10 from Coptis chinensis Franch and 20 from Phellodendron amurense Rupr., Peaks 3, 4, 6, 9, 11 and 12 came from them together. Peak 7 presented in the chromatograms of the herbs except Gardenia jasminoides Ellis. By comparison of the retention time, the on-line UV spectra and MS spectra, 11 peaks were identified as 5 (geniposide), 9 (jatrorrhizine), 10 (coptisine), 11 (palmatine), 12 (berberine), 13 (baicalin), 15 (oroxin A), 17 (wogonoside), 19 (baicalein), 20 (obaculactone), 21 (wogonin), then eight of them were quantified by HPLC-UV. CONCLUSION: The method could represent the characteristics of Huanglianjiedu decoction, and it could be used to evaluate the quality and quantity of Huanglianjiedu decoction. It distinguished between Coptis chinensis Franch and Phellodendron amurense Rupr. by HPLC for the first time.

Separation and identification of Aconitum alkaloids and their metabolites in human urine.

To study the safety of Aconitum medicinal herbs in clinic and identify Aconitum alkaloids poisoning in forensic medicine, Aconitum alkaloids and their metabolites were separated and identified in human urine by liquid chromatography-electrospray ionization-multi-stage mass spectrometry (LC-ESI-MS(n)) and chemical pathway of metabolism was investigated. The alkaloids and their metabolites in the urine sample were extracted with solid-phase cartridges and separated by HPLC with acetonitrile-water-formic acid (40:60:0.5) mobile phase. Structures of five metabolites and three parent Aconitum alkaloids were identified with multi-stage mass spectrometry data through comparison with authentic substances as aconitine (M(1)), mesaconitine (M(2)), hypaconitine (M(3)), benzoylaconine (M(4)), benzoylmesaconine (M(5)), benzoylhypaconine (M(6)), 16-O-demethylaconitine (M(7)) and 16-O-demethylhypaconitine (M(8)), respectively. Among them, M(8) was identified and reported for the first time. Metabolic pathways of Aconitum alkaloids in human body were proposed.

[Study on metabolites on aconitine in rabbit urine].

AIM: To identify the main metabolites of aconitine in the urine of rabbits. METHODS: After oral administration of aconitine (5 mg.kg-1), the urine of male rabbits was collected and extracted by solid phase extraction and analyzed by liquid chromatography-ion trap mass spectrometry. RESULTS: Aconitine and 4 metabolites were found in the rabbit urine. Their protonated molecular ions at m/z 632, m/z 604, m/z 590, m/z 500 and multistage fragment ions with neutral loss of 60 u, 32 u, 28 u and 18 u were monitored. Their relative concentration were M1 > Aconitine > M4 > M2 > M3. CONCLUSION: The metabolites M1-M4 were deduced as 16-O-demethylaconitine, benzoylaconine, 16-O-demethylbenzoylaconine and aconine, respectively.